Page 76 - Application Handbook - Liquid Chromatography
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LAAN-A-LC-E258







            Application                  High Performance Liquid Chromatography

            News                         Analysis of Sugars and Sugar Alcohols in Energy


                                         Drink by Prominence-i with Differential Refractive
            No.L481                      Index Detector





            Sugars and sugar alcohols display almost no ultraviolet
            absorption, and are therefore typically detected using a
            differential refractive index detector or evaporative light   uRI
            scattering detector. By using a ligand exchange column for   80                   n Peaks
            sugar analysis, it is possible to distinguish among the different                  1. maltose
            isomers based on the position of the hydroxyl group in the   70   1                2. glucose
            chair conformation of glucose and fructose for example. In               4         3. fructose
                                                                                               4. erythritol
            other words, the hydroxyl group of the sugar and the metal   60           5        5. mannitol
            ion of the stationary phase form a complex, making it               2  3           6. sorbitol
            possible to achieve separation due to the difference in the   50              6
            strength of the complex formation. Also, maintaining a   40
            column temperature of 80 °C suppresses sugar anomer
            separation and peak dispersion, thereby achieving good   30
            separation of adjacent peaks.
            The new Prominence-i integrated high-performance liquid   20
            chromatograph can be connected to the RID-20A differential
            refractive index detector. The column oven, which can   10
            accommodate a 30 cm column and maintain temperature
            control up to 85 °C, therefore supports applications that   0
            require a long column.
            In Application News No. 467, we introduced an example of   0  5     10    15    20    25   min
            analysis of sugars in juice, in which the Prominence-i was
            connected to a differential refractive index detector. Here, we
            introduce an example of simultaneous analysis of sugars and   Fig. 1  Chromatogram of a Standard Mixture of Six Sugars
            sugar alcohols in an energy drink using the Prominence-i and   (10 g/L each, 10 µL Injected)
            RID-20A.
            n Analysis of a Standard Mixture of Six Sugars
            Sorbitol, xylitol, mannitol and erythritol are a type of sugar
            alcohol that because of their relative sweetness, are used as   uRI
                                                                  80
            sweeteners. When conducting simultaneous analysis of sugars                       n Peaks
            and sugar alcohols, a hydrophilic compound analytical column,   70                  1. maltose
            such as the SPR-Ca or SPR-Pb, is suitable along with the use of                     2. glucose
            a combination of the size exclusion and ligand exchange   60    1  2                3. fructose
            modes of analysis. Fig. 1 shows the results of analysis of a                        4. mannitol
                                                                                                5. xylitol
            standard solution of six sugar alcohol substances (10 g/L each   50  3              6. sorbitol
            of maltose, glucose, fructose, erythritol, mannitol and sorbitol)
            using the SPR-Ca column with a 10 µL injection. The analytical   40      4
            conditions are shown in Table 1.                                             5
            Fig. 2 shows the results of analysis of a standard solution of six   30        6
            sugar substances including sugar alcohols (10 g/L each of
            maltose, glucose, fructose, mannitol, xylitol, sorbitol) using a   20
            10 µL injection, and Table 2 shows the analytical conditions
            that were used. The SPR-Pb was used as the analytical column.  10
                         Table 1  Analytical Conditions            0
             Column      : Shim-pack SPR-Ca (250 mm L × 7.8 mm I.D., 8 µm)
             Mobile Phase   : Water
             Flowrate    : 0.6 mL/min                                0      10      20      30      40  min
             Column Temp.   : 80 °C
             Injection Volume  : 10 µL
             Detection   : RID-20A
                          Polarity +, Cell temp. 40 °C, Response 1.5 sec  Fig. 2  Chromatogram of a Standard Mixture of Six Sugars
                                                                      (10 g/L each, 10 µL Injected)
                         Table 2  Analytical Conditions
             Column      : Shim-pack SPR-Pb (250 mm L × 7.8 mm I.D., 8 µm)
             Mobile Phase   : Water
             Flowrate    : 0.6 mL/min
             Column Temp.   : 80 °C
             Injection Volume  : 10 µL
             Detection   : RID-20A
                          Polarity +, Cell temp. 40 °C, Response 1.5 sec
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